The goal of this project is to develop a new low-cost MALDI-TOF mass spectrometer that outperforms all existing MS-MS techniques currently employed with MALDI. In the proposed instrument multiple segments of fragment spectra are required to generate a complete fragment spectrum, each segment corresponding to a particular range of the ratio of fragment mass to precursor mass; but unlike earlier post-source decay (PSD) instruments, accurate fragment ion masses may be determined simultaneously for fragments present due to many of the precursor ions in the spectrum. Thus although 10-15 segments may be required to generate a complete fragment spectrum, 100 or more precursors can be fragmented without sacrificing sensitivity or mass accuracy. A pulse rate of 5 kHz is employed, allowing data to be acquired much faster than in existing TOF instruments typically limited to rates of 200 hz or less. The emphasis is on optimum performance in MS-MS mode, but resolving power and mass accuracy in reflector MS mode are comparable to the best currently available in commercial instruments. Either positive or negative ions can be measured in both MS and MS-MS modes. The unique features of the proposed instrument include simultaneous acquisition of fragment spectra from a large number of precursors, rapid and sensitive precursor and neutral loss scans with high resolving power and mass accuracy, and acquisition of multiplexed MS3 spectra. The design effort is focused on simplicity, reliability, and minimum cost consistent with high performance. A modular approach is employed where many of the major components are common to a family of systems. Common components include the sample plate handling system with motion control, the laser optics and controls, vacuum system, digitizer and computer, and several electronics modules. A novel method for calibrating fragment spectra is described based on inversion of the equation describing the flight time in a two-stage ion reflector to yield an "exact" equation relating fragment mass to flight time. An important initial effort is focused on validating this approach and determining the fragment mass accuracy that can be achieved. A major portion of the research effort is directed toward developing the software required for processing time-of-fight data to automatically generate fragment spectra from simultaneous measurements on multiple precursors. The software will be extended to generating precursor scans, neutral loss scans, and multiple reaction monitoring with LC separation, and to interpreting MS3 time-of-flight data. The system will be validated for applications to protein identification by MS-MS on tryptic peptides and for quantitative and qualitative measurements on complex lipid samples. The Human Proteome Organization has launched major initiatives focused on plasma, liver, and brain proteomics. The premise of these initiatives is that "A comprehensive, systematic characterization of circulating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancer and other diseases". Omenn, Proteomics 2004, 4, 1235-1240. Progress on these initiatives was presented at the most recent HUPO meeting (Long Beach 2006). While many positive results were presented, an inescapable conclusion based on the lack of agreement among results from different laboratories on identical samples must be that present technology falls woefully short of that required to achieve the stated goals. This project, together with improved separations interfaces being developed in a separate project, will provide essential tools for comprehensive proteomic measurements. [unreadable] [unreadable] [unreadable]